RESUMO
Many biologically active peptide secondary metabolites of bacteria are produced by modular enzyme complexes, the non-ribosomal peptide synthetases. Substrate selection occurs through an adenylation (A) domain, which activates the cognate amino acid with high fidelity. The recently discovered Aâ domain of an Anabaenopeptin synthetase from Planktothrix agardhii (ApnA A1) is capable of activating two chemically distinct amino acids (Arg and Tyr). Crystal structures of the Aâ domain reveal how both substrates fit into to binding pocket of the enzyme. Analysis of the binding pocket led to the identification of three residues that are critical for substrate recognition. Systematic mutagenesis of these residues created Aâ domains that were monospecific, or changed the substrate specificity to tryptophan. The non-natural amino acid 4-azidophenylalanine is also efficiently activated by a mutant Aâ domain, thus enabling the production of diversified non-ribosomal peptides for bioorthogonal labeling.
Assuntos
Oscillatoria/enzimologia , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Azidas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oscillatoria/química , Oscillatoria/metabolismo , Peptídeos Cíclicos/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
The combination of enzymatic modification and bioorthogonal click chemistry provides a powerful approach for site-specific labeling of different classes of biomolecules in vitro and even in cellular environments. Herein, we describe a chemoenzymatic method to site specifically label 5'-capped model mRNAs independent of their sequence. A trimethylguanosine synthase was engineered to introduce alkyne, azido, or 4-vinylbenzyl moieties to the 5'-cap. These functional groups were then used for labeling using typical click reactions, such as the azide-alkyne cycloaddition or the tetrazine ligation.
Assuntos
Química Click/métodos , Metiltransferases/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/química , Reação de Cicloadição , Corantes Fluorescentes/química , Metiltransferases/genética , Estrutura Molecular , Engenharia de Proteínas , RNA Mensageiro/metabolismoRESUMO
Bioorthogonal click reactions are powerful tools to specifically label biomolecules in living cells. Considerable progress has been made in site-specific labeling of proteins and glycans in complex biological systems, but equivalent methods for mRNAs are rare. We present a chemo-enzymatic approach to label the 5' cap of eukaryotic mRNAs using a bioorthogonal photoclick reaction. Herein, the N7-methylated guanosine of the 5' cap is enzymatically equipped with an allyl group using a variant of the trimethylguanosine synthaseâ 2 from Giardia lamblia (GlaTgs2). To elucidate whether the resulting N (2)-modified 5' cap is a suitable dipolarophile for photoclick reactions, we used Kohn-Sham density functional theory (KS-DFT) and calculated the HOMO and LUMO energies of this molecule and nitrile imines. Our in silico studies suggested that combining enzymatic allylation of the cap with subsequent labeling in a photoclick reaction was feasible. This could be experimentally validated. Our approach generates a turn-on fluorophore site-specifically at the 5' cap and therefore presents an important step towards labeling of eukaryotic mRNAs in a bioorthogonal manner.
RESUMO
Trimethylguanosine synthase from Giardia lamblia (GlaTgs2) naturally catalyzes methyl transfer from S-adenosyl-L-methionine (AdoMet) to the exocyclic N(2) atom of the 5'-cap--a hallmark of eukaryotic mRNAs. The wild-type enzyme shows substrate promiscuity and can also use the AdoMet-analog AdoPropen for allyl transfer. Here we report on engineering GlaTgs2 to enhance the activity on AdoPropen. A mutational analysis, involving an alanine scan of 10 residues located around the active site, was performed. Positions V34 and S38 were identified as mutational hot spots and analyzed in greater detail by testing NNK libraries. Kinetic analysis and thermostability measurements revealed V34A as the best variant of GlaTgs2, with a â¼10-fold improved specificity for AdoPropen. Double mutants did not yield additional improvements due to low catalytic efficiencies and thermal destabilization. Homologous Tgs enzymes from Homo sapiens and G. intestinalis were also investigated regarding their catalytic activity on AdoPropen. While neither the human wild-type (WT) enzyme nor any of its variants showed activity on AdoPropen, the homologue from G. intestinalis (GinTgs) was remarkably active on AdoPropen. Introducing the best substitution at the homologous position led to variant T34A with â¼40-fold higher specificity for AdoPropen than the original GlaTgs2 WT.
Assuntos
Giardia lamblia/enzimologia , Metiltransferases/metabolismo , Mutagênese Sítio-Dirigida , Proteínas de Protozoários/metabolismo , Alanina , Estabilidade Enzimática , Temperatura Alta , Metiltransferases/química , Metiltransferases/genética , Mutação , Proteínas de Protozoários/química , Proteínas de Protozoários/genéticaRESUMO
Chemo-enzymatic strategies provide a highly selective means to label different classes of biomolecules in vitro, but also in vivo. In the field of RNA, efficient labeling of eukaryotic mRNA with small organic reporter molecules would provide a way to detect endogenous mRNA and is therefore highly attractive. Although more and more bioorthogonal reactions are being reported, they can only be applied to chemo-enzymatic strategies if a suitable (i.e., click compatible) modification can be introduced into the RNA of interest. We report enzymatic site-specific transfer of a 4-vinylbenzyl group to the 5'-cap typical of eukaryotic mRNAs. The 4-vinylbenzyl group gives access to mRNA labeling using the inverse electron-demand Diels-Alder reaction, which does not work with an enzymatically transferred allyl group. The 4-vinylbenzyl-modified 5'-cap can also be converted in a photoclick reaction generating a "turn-on" fluorophore. Both click reactions are bioorthogonal and the two step approach also works in eukaryotic cell lysate. Enzymatic transfer of the 4-vinylbenzyl group addresses the lack of flexibility often attributed to biotransformations and thus advances the potential of chemo-enzymatic approaches for labeling.